Cell-adhesion molecule uvomorulin is localized in the intermediate junctions of adult intestinal epithelial cells

Uvomorulin is a cell-adhesion molecule implicated in the compaction process of mouse preimplantation embryos and the aggregation of embryonal carcinoma cells. A rabbit antiserum against purified uvomorulin also reacts with epithelial cells of various adult tissues. In this study, we investigated the localization of uvomorulin on adult intestinal epithelial cells using electron microscopic analyses. Uvomorulin was shown to exhibit a highly restricted localization in the intermediate junctions of these cells. The results are discussed with respect to a possible adhesive function of uvomorulin on intestinal epithelial cells.     

Experimental manipulations of compaction and their effects on the phosphorylation of uvomorulin

Compaction of the eight-cell stage mouse embryo is a critical event in the generation of different cell types within the preimplantation embryo. Uvomorulin, a member of the cadherin family of cell adhesion molecules, is important during compaction and its phosphorylation increases early in the eight-cell stage, suggesting that this posttranslational modification may be important for compaction to proceed. We have assessed the importance of the phosphorylation of uvomorulin during compaction by preventing, reversing, or inducing adhesion prematurely. The only condition that affected the overall level of uvomorulin phosphorylation was the prevention of compaction through prolonged exposure of four-cell embryos to low Ca²⁻. This treatment reduced the level of uvomorulin phosphorylation in eight-cell embryos, and perturbed its localization to regions of cell-cell contact. Thus, whilst the phosphorylation of uvomorulin does not appear to regulate directly uvomorulin's adhesive function, it may be associated with the redistribution of uvomorulin during compaction. © 1996 Wiley-Liss, Inc.     

Reassortment of cells according to position in mouse morulae

Sixteen-cell mouse morulae were disaggregated and blastomeres originally occupying outer or inner positions were separated. Outer, inner, or unsorted populations of blastomeres were labeled with either trinitrobenzene sulphonic acid (TNP) or fluorescein isothiocyanate (FITC) and individual blastomeres aggregated to unlabelled partially decompact eight- to ten-cell morulae. After up to 6 h in culture, the positions of the labelled blastomeres within the aggregates were examined. The combined results demonstrated that between 86 and 92% of outer cells remained on the surface of the aggregate and flattened into extensive polygonal shapes, whereas 76-77% of the inner cells had become engulfed by the host morula cells and retained their initial spherical shape. Using unsorted cells, 33-37% were internalised, which is compatible with the most recent estimates of the presence of six to eight inner cells at the 16-cell stage. The possibility that differential adhesiveness of the outer and inner cells is involved in the allocation of cells to the trophectoderm and inner cell mass of the blastocyst is discussed.     

Ultrastructural characteristics of fresh and frozen-thawed ovine embryos using two cryoprotectants

Cryopreservation of sheep embryos with ethylene glycol as a protectant appears to be more effective than glycerol, particularly at the morula stage, as has been demonstrated on the basis of in vitro and in vivo development rates after thawing. In this study we compare the ultrastructure of fresh morulae, thawed morulae, and blastocysts cryopreserved with either ethylene glycol or glycerol at the electron microscopic level, to look for cellular damage that could be responsible for proven differences in embryo survival after transfer. Embryos cryopreserved with glycerol showed unequal degrees of conservation even among blastomeres within a single embryo. In morulae, inner blastomeres were completely damaged, whereas external ones appeared to be intact. Both morulae and blastocysts cryopreserved with ethylene glycol showed a higher uniformity in blastomere conservation than embryos with glycerol. The most remarkable features in this experimental group were the presence of desmosomes following tight junctions between blastomeres and the presence of many microvilli on the outer surface of external blastomeres. These characteristics are similar in fresh embryos of the control group. Our results show that ethylene glycol protects membrane and cytoplasmic structures of embryonic cells from cryoinjury much better than glycerol. In vivo survival of embryos confirmed the ultrastructural observations. A limited permeability of glycerol would explain the observed ultrastructural differences in blastomere integrity, which depends on blastomere location and the differences between morulae and blastocysts. We conclude that the low reproductive yield after cryopreservation using glycerol can be attributed to the lack of protection of inner cells.     

Delay of polarization event increases the number of Cdx2-positive blastomeres in mouse embryo

During preimplantation mouse embryo development expression of Cdx2 is induced in outer cells, which are the trophectoderm (TE) precursors. The mechanism of Cdx2 upregulation in these cells remains unclear. However, it has been suggested that the cell position and polarization may play a crucial role in this process. In order to elucidate the role of these two parameters in the formation of TE we analyzed the expression pattern of Cdx2 in the embryos in which either the position of cells and the time of polarization or only the position of cells was experimentally disrupted. Such embryos developed from the blastomeres that were isolated from 8-cell embryos either before or after the compaction, i.e. before or after the cell polarization took place. We found that in the embryos developed from polar blastomeres originated from the 8-cell compacted embryo, the experimentally imposed outer position was not sufficient to induce the Cdx2 in these blastomeres which in the intact embryo would form the inner cells. However, when the polarization at the 8-cell stage was disrupted, the embryos developed from such an unpolarized blastomeres showed the increased number of cells expressing Cdx2. We found that in such experimentally obtained embryos the polarization was delayed until the 16-cell stage. These results suggest that the main factor responsible for upregulation of Cdx2 expression in outer blastomeres, i.e. TE precursors, is their polarity.     

Temporally specific involvement of cell surface beta-1,4 galactosyltransferase during mouse embryo morula compaction

Cell surface beta-1,4 galactosyltransferase (GalTase) is shown to mediate intercellular adhesions between embryonal carcinoma (EC) cells and specifically during late morula compaction in the preimplantation mouse embryo. Monospecific anti-GalTase IgG raised against affinity-purified bovine beta-1,4 GalTase recognizes F9 EC cell GalTase as judged by immunoprecipitation and inhibition of GalTase activity, as well as by immunoprecipitation of a single 52 kd metabolically labeled membrane protein. Anti-GalTase IgG inhibits cell adhesions between EC cells, dissociates compacted mouse morulae, and inhibits blastocyst formation. Anti-GalTase IgG specifically inhibits cell adhesions during late morula compaction, coincident with a peak of surface GalTase activity as determined by direct enzyme assay. On EC cells, GalTase activity can be proteolytically released from intact cells, and is localized by indirect immunofluorescence to areas of intercellular contact, consistent with its proposed role in cell adhesion. Beta-1,4 GalTase is the first cell adhesion molecule identified that participates during late morula compaction, subsequent to uvomorulin function.     

Identification of a putative cell adhesion domain of uvomorulin.

A rat monoclonal antibody (DECMA-1) selected against the murine cell adhesion molecule uvomorulin blocks both the aggregation of mouse embryonal carcinoma cells and the compaction of pre-implantation embryos. However, decompacted embryos eventually become recompacted in the presence of DECMA-1 and form blastocysts composed of both trophectoderm and inner cell mass. DECMA-1 also disrupts confluent monolayers of Madin-Darby canine kidney (MDCK) epithelial cells. DECMA-1 recognizes uvomorulin in extracts from mouse and dog tissues. Protease digestion of mouse and dog uvomorulin generated core fragments including one of 26 kd which reacted with DECMA-1. The same 26-kd fragment is recognized by anti-uvomorulin monoclonal antibodies which have been obtained from other laboratories and which dissociate MDCK cell monolayers and block the formation of the epithelial occluding barrier. This 26-kd fragment therefore seems to be involved in the adhesive function of uvomorulin.     

Synthesis and phosphorylation of uvomorulin during mouse early development.

The cell adhesion molecule, uvomorulin, is synthesised in both the 135 x 10(3) M(r) precursor and 120 x 10(3) M(r) mature forms on maternal mRNA templates in unfertilized and newly fertilized mouse oocytes. Synthesis on maternal message ceases during the 2-cell stage to resume later on mRNA encoded presumptively by the embryonic genome. Uvomorulin is detectable by immunoblotting at all stages upto the blastocyst stage, but shows variations in its total amount and processing with embryonic stage. Whilst only trace levels of phosphorylated uvomorulin are detectable in early and late 4-cell embryos, uvomorulin in 8-cell embryos is phosphorylated.     

Scanning microscopy of disaggregated and aggregated preimplantation mouse embryos

Summary Blastomeres isolated from 8-and 16-cell embryos (that is 1/8 and 1/16) show a smooth surface at their point of contact with other blastomeres and a microvillous free surface. Microvilli reappear completely on the smooth surface of 52% of 1/8 embryos and partially on 88% of 1/16 embryos if cultured in vitro for 6 h. When 2-to 8-cell embryos are aggregated to 8-cell embryos and forced apart after 1–3 h, the contact surface of the 8-cell embryos has become smooth. Fixed 8-cell embryos are also able to induce complete disappearance of microvilli on the contact surface of a living 8-cell embryo. Embryos having more than 8 cells do not induce complete disappearance of microvilli on the contact surface of 8-cell embryos. Aggregates of late morulae do not show complete disappearance of microvilli at their contact surfaces but rather a loosening of their peripheral blastomeres.Our results show that isolated 1/8 and 1/16 embryos tend to recover from regionalization, that the process of aggregation of embryos having 8 cells or less is similar to compaction and that embryos having more than 8 cells seem to aggregate by cell sorting. The processes of compaction, adhesion and reassortment are briefly discussed. We submit that blastomere regionalization, which depends on cell to cell contact, may be the spatial basis of embryonic regulation and of the inside-outside normal differentiation of early mouse embryos.     

Antibodies to a renal Na+/glucose cotransport system localize to the apical plasma membrane domain of polar mouse embryo blastomeres.

Mouse preimplantation embryos were examined for the cell surface expression of epitopes that cross-react with antibodies to a 75-kDa subunit of a purified porcine renal brush border Na+/glucose cotransport system. A Na+ cotransport system is hypothesized to reside in the apical plasma membrane domain of mouse polar blastomeres and to be associated with the induction of their apical-basal polarity. Western blot analysis showed that unfertilized oocytes as well as preimplantation embryos contain a cross-reacting antigen with an apparent molecular weight of about 75,000. Embryos and their isolated blastomeres were double-labeled and assayed by indirect immunofluorescence (IIF) for the expression of epitopes (visualized by labeling with rabbit antiserum or mouse monoclonal IgG to cotransporter followed by the appropriate rhodamine-conjugated second antibodies) and for the development of cell surface polarity (visualized by the apical restriction of fluoresceinated succinylated concanavalin A binding; FS Con A). IIF did not detect these epitopes until after the second cleavage when 4-cell embryos expressed low-to-moderate levels. Although epitopes were expressed on all surfaces of 4-cell blastomeres, some blastomeres expressed more epitopes on their apical surfaces than on their basolateral ones. All precompaction 8-cell embryos expressed epitopes, with expression being greater apically on some blastomeres. The level of expression appeared to reach a maximum on morulae and to decline on cavitating embryos. Assays performed on isolated blastomeres from postcompaction embryos showed that by the 16-cell stage epitope expression appeared to become restricted to FS Con A-labeled apical plasma membrane domains and was no longer evident on basolateral domains. This apparent apical restriction of epitope expression was confirmed by electron microscopic examination of immunogold-labeled isolated polar 16-cell blastomeres. These results demonstrate that preimplantation mouse embryos contain an antigen(s) that is immunologically and structurally similar to a 75-kDa renal Na+/glucose cotransporter. The onset of cell surface expression of this antigen precedes development of the stable polar phenotype.     

The segregation of inner and outer cells in porcine embryos follows a different pattern compared to the segregation in mouse embryos

The mammalian blastocyst consists of an inner cell mass (ICM) enclosed by the trophectoderm. The origin of these two cell populations lies in the segregation of inner and outer cells in the early morula. In the present study, the segregation of inner and outer cells has been studied in porcine embryos and is compared with segregation in mouse embryos. For this, nuclei of inner and outer cells were differentially labelled with two fluorochromes after partial complement-mediated lysis of the outer cells. In porcine and mouse embryos compaction and the first appearance of inner cells occur at different stages of development. In porcine embryos compaction was observed as early as the 4-cell stage, while in mouse embryos compaction occurred in the 8-cell stage. The first inner cells segregated in porcine embryos which were in the transition from four to eight cells and inner cells were added during two subsequent cell cycles. In mouse embryos inner cells segregated predominantly during the fourth cleavage division. From the results obtained we conclude that the segregation of inner and outer cells follows a different pattern in mouse and in porcine embryos.     

Cell specific loss of polarity-inducing ability by later stage mouse preimplantation embryos

The individual blastomeres of the preimplantation mouse embryo become polarized during the 8-cell stage. Microvilli become restricted to the free surface of the embryo and this region of the membrane shows increased labeling with FITC-Con A and trinitrobenzenesulfonate (TNBS). Previous studies have shown that this polarity develops in response to asymmetric cell-cell contact with stage specific induction competent blastomeres. In the present study, the ability of later stage embryos to induce 8-cell polarization has been investigated. Newly-formed, nonpolar 8-cell stage blastomeres (1/8 cells) were isolated, then aggregated with morulae, inner cell clusters (from morulae), blastocysts, or inner cell masses (ICM) and cultured for 8 hr. Aggregates were then assayed for polarity. The results show a hierarchy of inducing ability, with the ICM and IC cluster possessing greater activity than the morula and polar trophectoderm of the early blastocyst, while the mural trophectoderm shows very little inducing activity. Furthermore, the inducing ability of the polar trophectoderm decreases with complete expansion and hatching of the blastocyst. These results indicate that the ability to induce 8-cell blastomere polarization is retained by the embryo beyond the 8-cell stage and that this ability is lost with further differentiation.     

Allocation of cells to inner cell mass and trophectoderm lineages in preimplantation mouse embryos

Inner cell mass (ICM) and trophectoderm cell lineages in preimplantation mouse embryos were studied by means of iontophoretic injection of horseradish peroxidase (HRP) as a marker. HRP was injected into single blastomeres at the 2- and 8-cell stages and into single outer blastomeres at the 16-cell and late morula (about 22- to 32-cell) stages. After injection, embryos were either examined immediately for localization of HRP (controls) or they were allowed to develop until the blastocyst stage (1 to 3.5 days of culture) and examined for the distribution of labeled cells. In control embryos, HRP was confined to one or two outer blastomeres. In embryos allowed to develop into blastocysts, HRP-labeled progeny were distributed into patches of cells, showing that there is limited intermingling of cells during preimplantation development. A substantial fraction of injected blastomeres contributed descendants to both ICM and trophectoderm (95, 58, 44, and 35% for injected 2-cell, 8-cell, 16-cell, and late morula stages, respectively). Although more than half of the outer cells injected at 16-cell and late morula stages contributed descendants only to trophectoderm (53 and 63%, respectively), some outer cells contributed also to the ICM lineage even at the late morula stage. Although the mechanism for allocation of outer cells to the inner cell lineage is unknown, our observation of adjacent labeled mural trophectoderm and presumptive endoderm cells implicated polarized cell division. This observation also suggests that mural trophectoderm and presumptive endoderm are derived from common immediate progenitors. These cells appear to separate into inner and outer layers during the fifth cleavage division. Our results demonstrate the usefulness of HRP as a cell lineage marker in mouse embryos and show that the allocation of cells to ICM or trophectoderm begins after the 2-cell stage and continues into late cleavage.     

Spatial distribution of blastomeres is dependent on cell division order and interactions in mouse morulae

Spatial distribution of blastomeres was examined in 16- to 30-cell morulae obtained from aggregates of 1/2----1/2 and 1/2----2/4 blastomeres. The advanced blastomeres (2/4) contributed disproportionately more inner cells while there was a corresponding decline in the contribution from the delayed blastomere (1/2) so that a balance between the total number of inner and outer cells was retained. There was, however, no marked change in the relative number of outer cells. It is suggested that once formed, the inner more adhesive cells divide relatively faster than the outer cells whose behaviour is dictated by the inner cells. The outer less adhesive cells spread over the inner cells; cell spreading is incompatible with division. The degree to which cell spreading and retardation of division of outer cells occurs may be dictated by the number of inner cells present at any one time and this partly determines the entry of further cells inside. The suggested mechanism for cell allocation is highly flexible and, indeed, essential to encompass the wide variety of patterns of cell interactions and distribution observed in morulae. It is also proposed that the retardation of division of outer cells may trigger differentiation of trophectoderm by inducing endoreduplication and the blastomeres delayed from dividing for the longest period of time may mark down the abembryonic pole and establish the embryonic-abembryonic axis.     

The morula-blastocyst transition in two Old World primates: the baboon and rhesus monkey

Baboon and rhesus monkey embryos demonstrate compaction during the morula stage, although not all blastomeres participate simultaneously. During this process the outer adherent cells develop progressively more extensive apical junctional complexes. Several spaces appear between blastomeres before formation of a single cavity, and assortment of inner cell mass and trophoblast cells is less rapid and less precise in primates than in rodents. Nonhuman primates should prove appropriate for studies of individual aberrant blastomeres during blastocyst formation.     

A quantitative analysis of cell allocation to trophectoderm and inner cell mass in the mouse blastocyst

The allocation of cells to the trophectoderm and inner cell mass (ICM) in the mouse blastocyst has been examined by labelling early morulae (16-cell stage) with the short-term cell lineage marker yellow-green fluorescent latex (FL) microparticles. FL is endocytosed exclusively into the outside polar cell population and remains autonomous to the progeny of these blastomeres. Rhodamine-concanavalin A was used as a contemporary marker for outside cells in FL-labelled control (16-cell stage) and cultured (approximately 32- to 64-cell stage) embryos, immediately prior to the disaggregation and analysis of cell labelling patterns. By this technique, the ratio of outside to inside cell numbers in 16-cell embryos was shown to vary considerably between embryos (mean 10.8:5.2; range 9:7 to 14:2). In cultured embryos, the trophectoderm was derived almost exclusively (over 99% cells) from outside polar 16-cell blastomeres. The origin of the ICM varied between embryos; on average, most cells (75%) were descended from inside nonpolar blastomeres with the remainder derived from the outside polar lineage, presumably by differentiative cleavage. In blastocysts examined by serial sectioning, polar-derived ICM cells were localised mainly in association with trophectoderm and were absent from the ICM core. In nascent blastocysts with exactly 32 cells an inverse relationship was found between the proportion of the ICM descended from the polar lineage and the deduced size of the inside 16-cell population. From these results, it is concluded that interembryonic variation in the outside to inside cell number ratio in 16-cell morulae is compensated by the extent of polar 16-cell allocation to the ICM at the next division, thereby regulating the trophectoderm to ICM cell number ratio in early blastocysts.     

Of mice, frogs and flies: generation of membrane asymmetries in early development

Embryonic development begins with cleavage of the fertilized egg. Cleavage comprises two major processes: cytokinesis and formation of a polarized epithelial cell layer. The focus of this review is comparison of the generation of membrane polarity during embryonic cleavage in three different developmental model systems. In mammalian embryos, as exemplified by analysis of the mouse, generation of distinct membrane domains is uncoupled from cleavage divisions and is initiated in a specific developmental phase, called compaction. In Xenopus laevis embryos, generation of polarized blastomeres occurs simultaneously with cytokinesis. The origin of specific membrane domains of X. laevis polar blastomeres, however, can be traced back to oogenesis. Finally, in Drosophila melanogaster, generation of polarized cells occurs at cellularization. The relevance of cell adhesion, cell junctions and cytocortical scaffolds will be discussed for each of the model systems. Despite enormous morphologic differences, the three models share many common features; in particular, many important molecular interactions are conserved.